In order to reach the oocyte and fertilize it, sperm must not only be vigorously motile, but must also modulate flagellar beating patterns appropriately. They must hyperactivate, which involves switching to high-amplitude, asymmetrical beating, as evidenced by the failure to fertilize of CatSper null mutant mouse sperm that cannot hyperactivate. Furthermore, there is evidence that sperm respond to chemotactic factors in order to direct their movement toward the oocyte. Although Ca2+ signaling is involved in both processes, the relationship of hyperactivation to chemotaxis is a mystery. Sperm become hyperactivated in the sperm reservoir in the lower oviduct before the time of ovulation and thus very likely before exposure to chemotactic signals from the egg mass. Our overall working hypothesis is that hyperactivation is modulated to direct sperm toward the oocyte. The objective of this R03 pilot project is to gain solid evidence for modulation of hyperactivation. Aim 1: To determine the flagellar movement patterns of sperm in the oviduct. The relationship of hyperactivation to chemotaxis will be studied for the first time in vivo using transillumination of oviducts of mated mice to analyze the movements of sperm as they escape from the reservoir in the lower oviduct and move toward oocytes. The symmetry and direction of asymmetrical bends will be evaluated. Aim 2: To distinguish the effects of Ca2+ influx through CatSper channels from those of Ca2+ release from redundant nuclear envelope (RNE) stores. CatSper channels are confined to the plasma membrane of the principal piece of the flagellum and the RNE is located at the base of the flagellum. Activation of CatSper channels produces deep pro-hook bends (in the same direction as the hook of the mouse sperm head), whereas stimulation of Ca2+ release from the RNE induces deep anti-hook bends. Switching from deep pro-hook to deep anti-hook bends could serve to adjust the course of sperm toward the oocyte. Ca2+ imaging and proteomic approaches will be used to distinguish the responses of sperm. Aim 3: To test the effects of putative chemotactic signals on behavior of hyperactivated sperm. If evidence is obtained in support of the hypothesis, then we will seek to identify the sources of modulators of sperm motility and further elucidate the cell signaling pathways that modulate sperm motility. Findings should inform efforts to treat infertility and to develop contraceptive methods. PUBLIC HEALTH RELEVANCE: Approximately 7% of US couples reported that they did not get pregnant after a year without contraceptive use. Roughly half of the cases of infertility are attributed to a male factor, particularly low sperm quantity and/or motility. To fertilize, sperm must be able to hyperactivate and also to respond to chemotactic factors to direct their movement toward the egg. Nothing is known about the relationship of hyperactivation to chemotaxis. The objective is to elucidate this relationship, by analyzing behavior of sperm in the oviduct and using cell biological and proteomic approaches to determine how sperm movement is modulated to enable them to reach the egg.